In our Department, various attempts have been made in order to elucidate the mechanism of carcinogenesis by azo dyes using tissue culture system. In the present experiments, a total of 28 Donryu rats were fed with 4-dimethyl-aminoazobenzene(DAB) or 3'-methyl-4-dimethyl-aminoazobenzene (3'-Me-DAB) for 5-62 days. Five cell lines have been established from the liver tissues of rats fed by either of the above substance. Subsequently, several experiments were made to investigate the histological characteristics of the established cell lines. The results are summarized as follows: 1) Morphological features differed in the cell lines derived from rats fed by DAB and those from rats fed by 3'-Me-DAB. The former exhibited polygonal shape and formed pavement-like sheets. On the other hand, the latter showed oval shape, but did not form pavement-like sheets. 2) In chromosome analysis, the cell lines derived from rats fed by DAB exhibited the diploid number in contrast to those from rats fed by 3'-Me-DAB. 3) In rotation culture, 5 cell lines formed aggregates. The aggregates of the cell lines derived from rats fed by 3'-Me-DAB were much larger in size than those from rats fed by DAB. 4) Concerning branched-chain amino acid transaminase, cultured cells derived from rats fed by 3'-Me-DAB contained the isozymes I and Ⅲ, whereas cells from rats fed by DAB contained only the isozyme I. 5) With radioimmunoassay alpha-fetoprotein level in culture medium of 5 cell lines ranged between 5 to 50 ng/ml. 6) To examine tumor-producing capacity of 5 cell lines, the cells were inoculated into rats intraperitoneally or subcutaneously. All cell lines induced by 3'-Me-DAB gave rise to tumors, but 3 cell lines induced by DAB did not produce tumors. The recultured cells derived from ascites of rats that died of tumors were morphologically similar to the cells before inoculation.